Bottom Line:
Our results show that wild-type lamin A negatively modulates TGFbeta 2 levels in osteoblast-like U2-OS cells, while the R527H mutated prelamin A as well as farnesylated prelamin A do not, ultimately leading to increased secretion of TGFbeta 2.TGFbeta 2 neutralization rescues Akt/mTOR activation and the downstream transcriptional effects, an effect also obtained by statins or RAD001 treatment.Our results unravel an unexpected role of lamin A in TGFbeta 2 regulation and indicate rapamycin analogs and neutralizing antibodies to TGFbeta 2 as new potential therapeutic tools for MADA.

ABSTRACTTransforming growth factor beta (TGFbeta) plays an essential role in bone homeostasis and deregulation of TGFbeta occurs in bone pathologies. Patients affected by Mandibuloacral Dysplasia (MADA), a progeroid disease linked to LMNA mutations, suffer from an osteolytic process. Our previous work showed that MADA osteoblasts secrete excess amount of TGFbeta 2, which in turn elicits differentiation of human blood precursors into osteoclasts. Here, we sought to determine how altered lamin A affects TGFbeta signaling. Our results show that wild-type lamin A negatively modulates TGFbeta 2 levels in osteoblast-like U2-OS cells, while the R527H mutated prelamin A as well as farnesylated prelamin A do not, ultimately leading to increased secretion of TGFbeta 2. TGFbeta 2 in turn, triggers the Akt/mTOR pathway and upregulates osteoprotegerin and cathepsin K. TGFbeta 2 neutralization rescues Akt/mTOR activation and the downstream transcriptional effects, an effect also obtained by statins or RAD001 treatment. Our results unravel an unexpected role of lamin A in TGFbeta 2 regulation and indicate rapamycin analogs and neutralizing antibodies to TGFbeta 2 as new potential therapeutic tools for MADA.

Mentions:
To support the above reported involvement of Akt/mTOR pathway in the altered regulation of OPG and cathepsin K, we inhibited Akt activity, using MK2206 and mTOR, using the rapamycin analog RAD001 that impairs mTORC1 activity (Figure 4). While Akt activity was completely inhibited by MK2206, protein amount was increased (Figure 4A). Akt inhibition caused dysregulation of its downstream effectors, including P70S6K and S6RP, although with different effects in WT lamin A versus R527H-mutated lamin A-transfected cells (Figure 4A). Phosphorylation of P70S6K was increased in WT-transfected cells and decreased in R527H-transfected U2-OS, while S6RP phosphorylation was consistently inhibited in all MK2206-treated cells (Figure 4A). RAD001 minimally affected Akt activity, but elicited protein increase (Figure 4A), possibly due to a feedback mechanism. Complete inhibition of P70S6K and S6RP phosphorylation was observed in all RAD001-treated cells (Figure 4A).

Mentions:
To support the above reported involvement of Akt/mTOR pathway in the altered regulation of OPG and cathepsin K, we inhibited Akt activity, using MK2206 and mTOR, using the rapamycin analog RAD001 that impairs mTORC1 activity (Figure 4). While Akt activity was completely inhibited by MK2206, protein amount was increased (Figure 4A). Akt inhibition caused dysregulation of its downstream effectors, including P70S6K and S6RP, although with different effects in WT lamin A versus R527H-mutated lamin A-transfected cells (Figure 4A). Phosphorylation of P70S6K was increased in WT-transfected cells and decreased in R527H-transfected U2-OS, while S6RP phosphorylation was consistently inhibited in all MK2206-treated cells (Figure 4A). RAD001 minimally affected Akt activity, but elicited protein increase (Figure 4A), possibly due to a feedback mechanism. Complete inhibition of P70S6K and S6RP phosphorylation was observed in all RAD001-treated cells (Figure 4A).

Bottom Line:
Our results show that wild-type lamin A negatively modulates TGFbeta 2 levels in osteoblast-like U2-OS cells, while the R527H mutated prelamin A as well as farnesylated prelamin A do not, ultimately leading to increased secretion of TGFbeta 2.TGFbeta 2 neutralization rescues Akt/mTOR activation and the downstream transcriptional effects, an effect also obtained by statins or RAD001 treatment.Our results unravel an unexpected role of lamin A in TGFbeta 2 regulation and indicate rapamycin analogs and neutralizing antibodies to TGFbeta 2 as new potential therapeutic tools for MADA.

ABSTRACTTransforming growth factor beta (TGFbeta) plays an essential role in bone homeostasis and deregulation of TGFbeta occurs in bone pathologies. Patients affected by Mandibuloacral Dysplasia (MADA), a progeroid disease linked to LMNA mutations, suffer from an osteolytic process. Our previous work showed that MADA osteoblasts secrete excess amount of TGFbeta 2, which in turn elicits differentiation of human blood precursors into osteoclasts. Here, we sought to determine how altered lamin A affects TGFbeta signaling. Our results show that wild-type lamin A negatively modulates TGFbeta 2 levels in osteoblast-like U2-OS cells, while the R527H mutated prelamin A as well as farnesylated prelamin A do not, ultimately leading to increased secretion of TGFbeta 2. TGFbeta 2 in turn, triggers the Akt/mTOR pathway and upregulates osteoprotegerin and cathepsin K. TGFbeta 2 neutralization rescues Akt/mTOR activation and the downstream transcriptional effects, an effect also obtained by statins or RAD001 treatment. Our results unravel an unexpected role of lamin A in TGFbeta 2 regulation and indicate rapamycin analogs and neutralizing antibodies to TGFbeta 2 as new potential therapeutic tools for MADA.